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United States Patent |
6,025,141
|
Hu
|
February 15, 2000
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Immunofluorescence assay for the detection of antibodies using
recombinant antigens in insoluble form
Abstract
The present invention relates to the use of insoluble forms of recombinant
proteins in a flow cytometric immunofluorescence assay for the detection
of given antibodies.
Inventors:
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Hu; Yu-Wen (Gloucester, CA)
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Assignee:
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The Canadian Red Cross Society (Ottawa, CA)
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Appl. No.:
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392794 |
Filed:
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September 12, 1995 |
PCT Filed:
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December 9, 1994
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PCT NO:
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PCT/CA94/00672
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371 Date:
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September 12, 1995
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102(e) Date:
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September 12, 1995
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PCT PUB.NO.:
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WO95/16040 |
PCT PUB. Date:
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June 15, 1995 |
Current U.S. Class: |
435/7.1; 424/184.1; 424/186.1; 424/187.1; 424/188.1; 424/189.1; 424/204.1; 424/806; 435/5; 435/7.92; 435/69.1; 435/69.3; 435/968; 435/971; 435/974; 435/975; 436/513; 436/800; 530/350; 530/403 |
Intern'l Class: |
G01N 033/53; G01N 033/537; G01N 033/567; C12P 021/06 |
Field of Search: |
424/880,801,188.1,208.1,184.1,264.1,186.1,187.1,189.1,806
530/403
536/23.72
436/513,800
435/7.1,7.2,7.21,339.1,338.35,7.92,5,69.1,69.3,968,971,975,974
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References Cited
U.S. Patent Documents
4118479 | Oct., 1978 | Prince et al.
| |
4129644 | Dec., 1978 | McAleer et al.
| |
4241175 | Dec., 1980 | Miller et al.
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4722840 | Feb., 1988 | Valenzuela et al.
| |
4734362 | Mar., 1988 | Hung et al.
| |
4752565 | Jun., 1988 | Folks et al.
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4870023 | Sep., 1989 | Fraser et al.
| |
4925784 | May., 1990 | Crowl et al.
| |
5041385 | Aug., 1991 | Kingsman et al.
| |
5156949 | Oct., 1992 | Luciw et al.
| |
5169784 | Dec., 1992 | Summers et al.
| |
5175098 | Dec., 1992 | Watanabe et al.
| |
5175099 | Dec., 1992 | Willis.
| |
5204259 | Apr., 1993 | Helting et al.
| |
Foreign Patent Documents |
0272858 | Jun., 1988 | EP | .
|
0307149 | Mar., 1989 | EP.
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Other References
Shoeman et al. "Comparison of Recombinant Human Immunodeficeiency Virus gag
Precursor and gag/env Fusion Proteins and a Synthetic env Peptide as
Diagnostic Reagents". Analytical Biochemistry, vol. 161, pp. 370-379,
1987.
Wagner et al. "Studies on processing, particle formation, and
immunogenicity of the HIV-1 gag gene product; a possible component of a
HIV vaccine", Arch Virology, vol. 127, pp. 117-137, 1992.
Gaskin et al. "Use of chemical cleavage active HIV-1 proteinase from a
fusion protein produced in the form of insoluble inclusion bodies",
Biochemical Society Transaction, vol. 20, No. 2, pp. 162S, May 1992.
J.M. Hofbauer et al Journal of Clinical Microbiology, Jan., 1988,
26:116-120, "Comparison of Western Blot (Immunoblot) Based on
Recombinant-Derived p41 With Conventional Test for Serodiagnosis of Human
Immunodeficiency Virus Infections".
L. Luo et al., Proc. Natl. Acad. Sci. USA, Nov., 1992, 89:10527-10531,
"Chimeric gag-V3 Virus-Like Particles of Human Immunodefiency Viorus
Induce Virus-Neutralizing Antibodies".
J.M. Sligh et al., A.J.C.P.,Feb., 1989, 91:210-214, "Flow Cytometric
Indirect Immunoflorescence Assay with High Sensitivity Specificity for
Detection of Anitbodies to Human Immunodeficiency Virus (HIV)".
|
Primary Examiner: Knode; Marian C.
Assistant Examiner: Lee; Datquan
Attorney, Agent or Firm: Browdy and Neimark
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATION
This application is a continuation-in-part of U.S. application Ser. No.
08/164,789, filed Dec. 10, 1993, now abandoned the content of which is
entirely incorporated herein by reference.
Claims
The embodiments of the invention in which an exclusive property or
privilege is claimed are defined as follows:
1. A method for detection of a disease state which comprises utilizing an
insoluble form of at least one recombinant protein in a flow cytometric
immunofluorescence assay, said method comprising:
(i) obtaining a sample of biological fluid within which the disease state
is to be detected;
(ii) adding a predetermined amount of said insoluble form of at least one
recombinant protein to said sample to form a mixture, wherein said
insoluble form being produced in particle form by expression in
baculovirus or in an expression system producing a particle form, and
wherein said insoluble form is the carrier and the antigen;
(iii) incubating said mixture for a period of time sufficient to permit
association between said insoluble form of at least one recombinant
protein and antibodies within said sample to form an incubated mixture;
(iv) washing said incubated mixture;
(v) adding a labelled antibody to the mixture of step (iv) and incubating
for a period of time sufficient for said labeled antibody to bind with
antibodies within said incubated mixture to form a labeled mixture;
(vi) washing said labeled mixture;
(vii) detecting IgM complexes formed in said washed labeled mixture using
flow cytometric immunofluorescence; and
(viii) correlating detection of IgM complexes with a disease state, wherein
an increase level of IgM over the control level is indicative of the
disease state.
2. The method of claim 1 wherein the recombinant protein is expressed in a
baculovirus.
3. The method of claim 1 wherein the disease state is selected from the
group consisting of HIV infection, hepatitis and HTLV infection.
4. The method of claim 3 wherein the disease is selected from the group
consisting of HIV-1 infection, HIV-2 infection, hepatitis B infection,
hepatitis C infection, HTLV-1 infection and HTLV-2 infection.
5. The method of claim 4 wherein the disease state is HIV-1 infection.
6. The method of claim 4 wherein the disease state is hepatitis B
infection.
7. The method of claim 3, wherein the disease state is HIV infection and
the said at least one recombinant protein comprises po197, gp160, or a
protein comprising SEQ ID NO:2, optionally fused to an amino acid sequence
of a selected fusion protein having a sequence selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; or mixtures
thereof, and variants thereof.
8. The method of claim 7 wherein a mixture of gag-p45, po197 and gp160 is
utilized as carrier and antigen.
9. The method of claim 7, wherein said recombinant protein further
comprises an amino acid sequence of SEQ ID NO:4 as the selected fusion
partner.
10. The method of claim 7, wherein said recombinant protein further
comprises an amino acid sequence of SEQ ID NO:6 as the selected fusion
partner.
11. The method of claim 7, wherein said recombinant protein further
comprises an amino acid sequence of SEQ ID NO:8 as the selected fusion
partner.
12. The method of claim 7, wherein the selected fusion partner has at least
one immunoreactive domain.
Description
FIELD OF THE INVENTION
The present invention relates to an assay for the detection of various
disease states which utilizes antigens in insoluble form and more
particularly, for the detection of HIV infection. The assay of the present
invention has a high sensitivity allowing for detection of antibodies to
the HIV virus in the "window" period.
BACKGROUND OF THE INVENTION
Enzyme immunoassays (EIA) and Western Blot assays (WB) have been routinely
used for detecting HIV infection for several years. The implementation of
these tests has significantly reduced the risk of transfusion-related HIV
infection. However, some recent studies based on the detection of HIV DNA
have demonstrated that some HIV infected individuals do not have any
detectable amount of anti-HIV antibodies when the approved tests are
performed. Data from several studies indicate that there is a "window
period" estimated to span from a few weeks to several months or even
several years between initial HIV infection and seroconversion. Cases of
post-transfusion HIV infection have been reported from seronegative blood
donors. This is assumed to be a consequence of donation during the
"window" period on the part of these donors.
To verify the presence of HIV-antibody in donors of reactive samples to
EIA, the Western Blot assay has been used for confirmatory testings
(Ulstrup, J. C. et al, Lancet i:1151-1152). In this assay, 6 to 9
characteristic bands indicating antibodies to HIV surface and core
antigens are observed if antibodies to HIV-1 proteins are present
(positive), and no bands if antibodies are absent (negative). However, a
significant proportion of EIA repeatedly reactive samples react only to
the HIV-1 gag-derived core proteins (p17, p24 and p55) on the WB test
(Kleinman, S., 1990, Arch. Pathol. Lab. Med. 114:298-303, Tribe D. E. et
al, 1988, J. Clin. Microbiol. 26:641-647). This reactivity does not meet
the definition of HIV-1 positive or negative in the criteria for WB tests
and as a result these samples are labelled as HIV-1 WB indeterminates.
Studies have established that 30 to 40 percent of EIA repeatedly positive
donors are HIV-1 WB indeterminates, a figure that is typical in North
America. Follow-up studies performed in regions of high prevalence of HIV
show that while 95 to 99 percent of these are not infected with HIV, the
remaining 1 to 5 percent of blood donors with HIV-1 WB indeterminate
results are true seroconverters, usually at an early stage of infection
(Busch, M. P. et al., 1991, Transfusion 31:129-137; Gallo D. et al, 1986,
J. Clin. Microbiol. 23:1049-1051). In order to optimize the safety of
transfusion, all donors with HIV-1 WB indeterminate results are deferred.
The majority of donors with HIV-1 WB indeterminate results undergo
needless anxiety, their deferral represents a significant loss of donors
and recipients are still worried about contamination of blood taken from
"window" period of seroconversion donors. A highly sensitive assay is thus
also desirable in order to properly classify these indeterminate results.
Partially purified disrupted virus is used as an antigen for most currently
licensed screening and confirmatory tests. Human cells are always used for
culturing the HIV-1 virus (Dodd, R. Y. and C. T. Fang, 1990, Arch. Pathol.
Lab. Med. 114:240-245). Recombinant proteins and synthetic peptides have
been recently licensed for screening tests (Busch M. P. et al., 1991,
Transfusion 31:129-137; Das P.C. et al., 1992, Trans Med 2:249-250;
Ramirez E. P., 1992, J. Clin. Microbiol. 30:801-805). In theory, these
antigens can provide more sensitive and definitive assays. However, most
recombinant proteins are produced in E. coli and denatured during the
purification and processing of the antigens. Also, a certain proportion of
donors still show cross-reactivity to HIV core antigens (such as antigen
p24), and sensitivity is limited to detecting very early HIV antibodies.
A serious drawback to the use of synthetic peptides is related to the fact
that in some HIV-1 infected patients and seroconverters in high risk
populations, the serum antibody titre is very low, or undetectable, either
because of complex formation between p24 and antibodies or the loss of
specific clones of antibody producing cells (Orsknov, L. B., Eur. J. Clin.
Microbiol. Inf. Dis. 8:614). Synthetic peptides, which in general only
cover one or two epitopes, do not efficiently detect such low titre
antibodies and furthermore have a limited ability to take on the natural
three dimensional structures of antigen.
An immunofluorescence assay (IFA) has recently been licensed as an
alternate confirmatory test for detecting HIV-1 antibodies (FDA
Memorandum, 1992, Summer:56-67). IFA is rapid, simple and inexpensive.
However, it is a subjective procedure requiring well trained personnel
(Ascher, M. S. 1990, Arch. Pathol. Lab. Med. 114:246-248). This is a
limitation for users and false positive and false negative results still
occur in the IFA tests because of cross-reactivity of some antibodies to
antigens on the human cells in which the HIV virus is cultured. Fixing of
the infected cells before incubation also has been shown to lead to false
positive and negative results (McHugh, T. M., 1986, Diagnos. Immunol.
4:233-240).
An assay based on immunofluorescence was recently developed to detect
antibodies to HIV-1 by using flow cytometry (FIFA) (Sligh, J. M., 1989,
Am. J. Clin. Path. 91:210-214). FIFA is a sensitive, quantitative test. In
a typical FIFA protocol, HIV-1 infected cells are used directly for the
test. However, false positive and false negative results may occur because
of the HIV-1 antibody cross-reactivity to the antigens (such as HLA) on
human cells in which the virus is cultured and used as antigens in FIFA.
Another concern is with biohazards caused by the infectious virus which is
a big limitation for users. Fixing the HIV-1 infected cells for
inactivation of the virus before incubation has shown to lead to higher
cross reactivity on the cells.
The present invention is directed to a flow cytometric immunofluorescence
assay (r-FIFA) using insoluble forms of recombinant proteins expressed in
an expression system such as the baculovirus system. In a preferred
embodiment, r-FIFA is used for the detection of the HIV virus infection.
Insoluble forms of recombinant HIV-1 proteins such as HIV-1 gag p45
protein, gag gp-41 chimeric proteins, HIV-1 precursor polyproteins pol 97
and gp160 are used as autologous carriers (in place of beads) and antigens
to detect HIV-1 antibodies using flow cytometry. The baculovirus
expression system has become a major recombinant protein production system
because of several advantages over bacterial and mammalian systems
including superior yield of recombinant protein, safety (baculovirus is
not infectious to humans) and fidelity of its products.
In sensitivity comparison between r-FIFA and currently licensed tests,
r-FIFA was found to be more sensitive, the average increase in sensivity
for early detection of HIV-1 infection being greater than 20 days. r-FIFA
has permitted the detection and quantification of HIV-1 specific IgG, IgM
and IgA antibodies during the window period. The use of HIV-1 recombinant
proteins in an immunofluorescence assay (r-FIFA) solves the problems of
antibody cross-reactivity to antigen on human cells and the biohazard
concern with the original FIFA.
SUMMARY OF THE INVENTION
In a broad embodiment, the invention relates to the use of an insoluble
form of at least one recombinant protein as a carrier and antigen in a
flow cytometric immunofluorescence assay for the detection of a disease
state wherein said protein reacts with an antibody present in said disease
state. In a preferred embodiment, the invention relates to the use of an
insoluble form of at least one recombinant protein as a carrier and
antigen in a flow cytometric immunofluorescence assay for the detection of
HIV.
BRIEF DESCRIPTION OF THE DRAWINGS
The file of this patent contains at least one drawing executed in color.
Copies of this patent with color drawing(s) will be provided by the Patent
and Trademark Office upon request and payment of the necessary fee.
The invention will be better understood through the following detailed
description of the preferred embodiment in conjunction with the
accompanying drawings in which:
FIGS. 1A-1C represent the construction of a recombinant baculovirus
containing the HIV-1 gag gene sequence coding for gag-45 protein. The
gag-45 coding region was isolated from the plasmid pHxB-2D (8). FIG. 1A; a
Cla1-Bgl II fragment was modified and subcloned into pUC19 by ligation
with two synthetic oligonucleotide linkers. FIG. 1B; the linker 1 (SEQ ID
NOS:9 and 10) contains a Bam H1 site and the missing sequence including
the translation initiation codon (ATG) at N-terminal of gag gene. The
linker 2 (SEQ ID NOS:11 and 12) creates a translation termination codon
(TAA) followed by a Bam H1 site. FIG. 1C; the Bam H1 fragment was isolated
from the pUC19-gag45 and inserted into the Bam H1 site of transfer vector
pAcYM1 (9). The recombinant transfer vector pAcYM1-gag 45 was used for
co-transfection of SF9 cells with wild type AcNpV DNA and then the
recombinant baculovirus gag45 gene was isolated to express the recombinant
protein gag45;
FIGS. 2A and 2B show the nucleotide sequence of HIV-1 gag protein p45 (SEQ
ID NO:1);
FIG. 3 is the amino acid sequence of HIV-1 gag protein p-45 (SEQ ID NO:2);
FIG. 4 is a schematic representation of the construction of recombinant
HIV-1 gag gp41 proteins. Three gag-gp 41 chimeric proteins were
constructed based on the truncated gag precursor p45 gene sequence. The
gp41 coding region A (nucleotides 7737-8090), 118 a.a.), B (nucleotides
7923-8264, 114 a.a.) and C (nucleotides 7737-8264, 176 a.a.) were
amplified by PCR with primers P.sub.1 (SEQ ID NO:13), P.sub.2 (SEQ ID
NO:14), p.sub.3 (SEQ ID NO:15) and p.sub.4 (SEQ ID NO:16) which contain a
Bgl II enzyme site at both ends and a stop codon TAA at the 3' end and
then inserted respectively into the Bgl II enzyme site at 3' end of
gag-p45 coding sequence in the recombinant plasmid pAcYm1 gag p45. The
chimeric DNAs were isolated and inserted into the Bam H1 cloning site of
AcNPV transfer vector plasmid pVL 1393. The recombinant viruses gag-gp
41A, gag-gp 41B and gag-gp 41C were isolated and purified after
co-transfection;
FIG. 5 is the nucleotide sequence (SEQ ID NO:3) and amino acid sequence of
chimeric protein A (SEQ ID NO:4);
FIG. 6 is the nucleotide sequence (SEQ ID NO:5) and amino acid sequence of
chimeric protein B (SEQ ID NO:6);
FIG. 7 is the nucleotide sequence (SEQ ID NO:7) and amino acid sequence of
chimeric protein C (SEQ ID NO:8); and
FIGS. 8A-8D represent the titration of HIV-1 seropositive plasma using
gag-p45 as the antigen in r-FIFA. FIG. 8A: a flow cytometric histograms of
the seropositive and seronegative plasma are presented showing HIV-1
specific antibody (IgG) positive signal (the line peaks) and the negative
signal (solid peak). Each of the two line peaks represent the duplicates
of the assay. The heavy overlapping of the duplicates indicates the high
reproducibility of r-FIFA. FIG. 8B: The median fluorescence intensity of
the HIV-1 positive sample (.largecircle.) and the negative sample were
plotted (.circle-solid.) over the dilutions (1:25 to 1:25 600). The median
fluorescence intensity ratio of HIV-1 positive sample to HIV-1 negative
sample (S/N) is highest at 1:25. C: The dilution of 1:25 was chosen for
r-FIFA to detect IgG (FIG. 8C) and IgM (FIG. 8D) antibodies (B-IgG and
B-IgM) to HIV-1 in a HIV-1 positive sample by double staining with goat
anti-human IgG FITC and IgM R-PE;
FIG. 9 represents early HIV-1 antibodies detection in the samples of BB1
anti-HIV-1 seroconversion panel D by r-FIFA using two different antigens.
The flow cytometric histograms show anti-HIV-1 antibody signals in those
samples (line peaks) and fluorescence signal (background) of the negative
sample as control (solid grey peak);
FIGS. 10A-10C represent detection of early HIV-1 antibodies (IgG and IgM)
using r-FIFA in the samples from three individuals (PHL-A, (FIG. 10A),
PHL-B, (FIG. 10B), PHL-C (FIG. 10C) who were infected but seronegative in
the first bleed (upper row) by recently licensed screening tests. The
numbers under IgG and IgM indicate the s/c value of the samples; 1.0 or
greater is considered positive;
FIGS. 11A-11H represent antibody response during the window period of HIV-1
infection. In this kinetic analysis of IgM and IgG antibody production the
results are expressed by median fluorescence intensity ratio of sample to
cut-off (s/c) value (left axis). The broken line represents the antibody
s/c value level of 1.0. The s/c value on this line or higher is considered
positive. The right axis represents the s/c (s/co) value of HIV-1
antigens. A typical primary immune response was found in panels (FIG.
11A), K (FIG. 11C), D (FIG. 11E) and R (A) (FIG. 11G). The immune response
pattern in panels E (FIG. 11B), H (FIG. 11D), P (FIG. 11F) and Q (B) (FIG.
11H) is different from the pattern seen in (FIGS. 11A, 11C, 11E and 11G).
IgG is the dominant antibody and remains at low level for a long period.
(.largecircle.) represents IgM antibody to HIV-1, (.circle-solid.)
represents IgG antibody to HIV-1 and (.tangle-soliddn.) represents HIV-1
antigen;
FIG. 12 represents the kinetics of the immune response during the window
period of HIV-1 infection. The fluorescence histograms of samples in BB1
panel K represent IgG, IgM and IgA antibody production showing the phases
of primary response at a very early stage of HIV-1 infection. IgA antibody
in samples K1 and K2 are significantly stronger than the negative control,
but they are still negative by our criteria because the s/c value is
higher for IgA than for IgG;
FIGS. 13A and 13B represent antibody detection using antigen gag-gp41-C
(p45-cc, FIG. 13A) and gag-p45 (p45, FIG. 13B) in r-FIFA. FIG. 13A: the
sample tested had weak antibodies to HIV-1 core (45) and strong antibodies
to gag-gp41 (cc) by Western Blot (IgG only).
FIG. 13B: the antigen gag-p45 (45) and antigen gag-gp41 (cc,B) were
visualized by Coomassie blue staining of the polyacrylamide gel. The
r-FIFA results (FIGS. 13A and 13B) are shown to be concordant with Western
Blot results;
FIGS. 14A-14C represent HIV-1 early antibody (IgG) detection by using the
gag-p45 antigen and the antigen chimeric gag-gp41-C (p45-cc) in r-FIFA.
The results shown indicate that chimeric antigen gag-gp41-C (p45-cc)
detects the antibodies earlier and with a stronger signal than the antigen
gag-p45 does during the early stage of HIV-1 infection FIGS. 14A-14C
represent data obtained from FIGS. 11C, 11A and 11G, respectively.
FIG. 15 represents antibody response to HIV-1 RT precursor pol97
polyprotein tested by r-FIFA during early HIV-1 infection in the
anti-HIV-1 seroconversion performance panel J(BB1). The axises represent
the s/co value of HIV-1 antigens (right) and the sample to cut-off ratio
(s/c) of antibody to pol97 (left). The broken line represents the antibody
s/c value of 1.0; and
FIG. 16 represents the detection of antibodies to HIV-1 gag-p45 particles
by indirect immunofluorescence. The HIV-1 antibody bound gag-p45 particles
were stained with goat antihuman IgG FITC and IgM RPE conjugates showing
the HIV-1 specific antibodies IgG (green fluorescence) and IgM (orange
yellow fluorescence).
DESCRIPTION OF THE PREFERRED EMBODIMENT
The present invention relates to the use of an insoluble recombinant
protein in a flow cytometric immunofluorescence assay, hereinafter
referred to as r-FIFA, for the detection of antibodies. In the examples
that follow, the use of insoluble forms of the HIV-1 gag precursor p55,
termed p45 protein, which includes p17, p24 and part of p16 and chimeric
particles of gag-gp41 fusion proteins; the gp160 and pol97 proteins for
the early detection of HIV as well as the hepatitis B core antigen for the
detection of hepatitis B is described.
Recombinant proteins for the present invention are prepared in the
baculovirus expression system since the baculovirus expression system has
several advantages over bacterial and mammalian systems. These include a
superior yield of recombinant protein, safety and fidelity of the
products. Glycosylation, myristolyation, proteolytic processing and other
post-translational modifications that occur in baculovirus expressed
proteins are similar or identical to the native HIV-1 proteins and to
HIV-1 proteins derived from mammalian cell culture systems.
When expressed in insect cells, baculovirus encoded rp45 exhibits two
molecular forms, an insoluble particle form and a soluble protein. The
particle form of gag protein consists of a membrane-enveloped corelike
particle released into the medium by budding at the plasma membrane. Both
of the two forms of gag rp45 are myristoylated (Mervis, R. J., 1988, J.
Virol. 62:3993-4002). HIV-1 gag particle is 100 to 120 nm in diameter. It
is a good carrier for inserting immunoreactive domains of other proteins,
such as neutralizing epitopes of gp120 by using recombinant DNA
techniques, thereby extending the immunoreactivity spectrum of this gag
protein.
Standard performance panels obtained from BBI (Boston Biomedica Inc.) were
used for the evaluation of r-FIFA. These performance panels are made
available to enable manufacturers and users to test and assess their
anti-HIV-1 test systems, especially with regard to specificity,
reproducibility and sensitivity. BBI provides comprehensive data for
comparative analysis. Each set of BBI panels includes 6 to 14 aliquots
assembled from a repository of frozen sera or plasma units. An anti-HIV-1
low titer performance panel (PRB-104), anti-HIV-1 seroconversion panels
D(PRB 904), E(PRB 905), H(PRB-908), J(PRB 910), K(PRB-911), P(PRB-916),
Q(PRB-917), R(PRB-918) and three HIV-1 seroconversion specimens provided
by PHL were tested and analyzed by r-FIFA. To confirm the specificity of
r-FIFA, 295 plasma or serum samples from random blood donors, 105 Western
Blot positive samples and 138 E1A reactive, Western Blot indeterminate
samples from The Canadian Red Cross Society, National Testing Laboratory
were also tested.
EXAMPLE 1
Construction of Recombinant Baculoviruses for the Expression of HIV-1 Gag
p45 and Chimeric gag-gp41 Proteins
Recombinant baculoviruses were constructed to express HIV-1 gag p45 and
chimeric gag-gp41 proteins gag-gp41 A, gag-gp41 B and gag-gp41 C. The gag
p45 coding region (including intact p17, p24 and part of p16 coding
sequences) was isolated from the plasmid pHxB-2D (Ratner et al., 1985,
Nature 313, 277-284). A Clal-Bgl II fragment was modified and subcloned
into pUC19 by using synthetic oligonucleotide linkers. Linker 1 contains a
Bam H1 site and the missing sequence including the translation initiation
codon (ATG) at the N-terminal of the gag gene. Linker 2 created a
translation termination codon (TAA) followed by a Bam H1 site. The Bam H1
fragment was isolated from pUC19-gag p45 and inserted into the Bam H1 site
of transfer vector pAcYM1 as illustrated in FIG. 1. The recombinant
plasmid pAcYM1-gag p45 was used for co-transfection of SF9 cells with wild
type AcNPV DNA and then the recombinant baculovirus gag-p45 was isolated
to express the recombinant protein gag p45. Three gag-gp41 chimeric
proteins were constructed and are referred to as gag-gp41 A, gag-gp41 B
and gag-gp41 C and represented in schematic form in FIG. 4. The gag coding
sequence in the three constructions is the same as gag p45. The gp41
coding sequences A (nucleotides 7737-8090, 118 a.a.), B (nucleotides
7923-8264, 114 a.a), and C (nucleotides 7737-8264 176 a.a.) were inserted
respectively at the Bgl II site at the end of the gag-p45 gene. The
nucleotide and amino acid sequences of gag-p45 (SEQ ID:1 A and SEQ ID:2
respectively) are provided in FIGS. 2 and 3. The nucleotide and amino acid
sequences of the three chimeric proteins are provided in FIGS. 5, 6 and 7
(SEQ ID:3 and 4 for Protein A, SEQ ID:5 and 6 for protein B and SEQ ID:7
and 8 for protein C). The chimeric DNAs were isolated and inserted into
the Bam H1 cloning site of transfer vector plasmid pVL 1393 and
cotransfection was performed using the BaculoGold system (Pharminogen).
Recombinant viruses were isolated as chimeric gag-gp41 A, gag-gp41 B and
gag-gp41 C.
Construction of Recombinant HIV-1 Gag/Env Chimeric Proteins
Three gag/env chimeric proteins were constructed and were referred to as
chimeric A, B, and C as shown in FIG. 4. The gag DNA sequence in the three
proteins was the same. That sequence contained a 5' truncated HIV-1 gag
protein (p45) sequence excluding the TAA termination codon. In addition,
chimeric A contained 118 a.a. env sequences (nucleotides 7737-8090 SEQ
ID:3). Chimeric B contained 114 a.a. env sequences (nucleotides 7923-8264
SEQ ID:5) and chimeric C contained 176 a.a. env sequences (nucleotides
7737-8264 SEQ ID:7). The chimeric DNA was inserted into the Bam H1
restriction site of plasmid pVL1393 and cotransfection was performed using
the BaculoGold transfection system. The nucleotide sequences of the three
chimeric proteins are provided in FIGS. 5, 6 and 7.
a) Amplification of HIV-1 env Regions
About 100 ng of HIV-1 plasmid DNA (pHxB-20) was used in the polymerase
chain reaction (PCR). Amplification was performed using primers p1 and p2
to amplify the env region of chimeric A. Primers p3 and p4 were used to
amplify the env region of chimeric B and primers p1 and p4 were used to
amplify the env region of chimeric C as shown in FIG. 4. PCR was performed
using the Perkin Elmer Cetus amplification kit and their cycler (Gene A mp
PCR System 9600). The amplification reaction was denatured by heating to
95.degree. C. for 20 seconds, then annealed at 68.degree. C. for 15
seconds and extended at 72.degree. for 45 seconds. A total of 30 cycles
were performed.
b) Cloning and Bgl II Digestion
The PCR product of each of the env sequences was inserted into the TA
cloning vector using the manufacturer's procedures (TA cloning kit,
Invitrogen). Using the same kit, transformation of E. coli competent cells
was performed and recombinant white colonies were picked up and grown in
LB medium. DNA was extracted from the cells using published procedures and
was used for digestion. This DNA was digested to completion with Bgl II
(Gibco BRL), which incises twice in the plasmid at each of the two primers
and hence releases the PCR product and introduces Bgl II sticky ends. This
DNA was then ligated to the Bgl II cut pAcyM1 (at position 1310
nucleotides) vector, which contains the truncated HIV-1 gag DNA sequence.
The ligated DNA was then introduced into E. coli competent cells and
recombinant white colonies were grown in LB media. This DNA was extracted
and used for Bam H1 digestion.
c) Barn H1 Digestion and Cloning Into pVL1393
The plasmid pAcYM 1, which contained the recombinant gag/env sequences, was
digested with Bam H1 (Gibco BRL) in order to release the entire insert.
This DNA was then ligated to Bam H1 cut pVL1393 (BaculoGold transfection
vector). After ligation and transformation of E. coli competent cells,
white colonies were grown in LB and DNA was extracted and purified.
d) Cotransfection
An equivalent of 5 .mu.g of recombinant pVL1393 was used in the
cotransfection procedure. This procedure was performed as recommended by
the manufacturer (BaculoGold transfection kit, Pharminogen).
It will be understood by a person skilled in the art that while the present
application only describes the preparation of three chimeric proteins,
other chimeric proteins could be made and used as well in the
immunofluorescence assay. Chimeric proteins containing conservative
regions and having at least an antigenic or immunoreactive domain or
epitope could also be used for the present invention.
EXAMPLE 2
Construction of Recombinant Baculovirus to Express HIV-1 gp160
A 5' end primer and 3' end primer having the following sequences:
5'end primer (SEQ ID NO: 17)
CGC TGA TCA ATG AGA GTG HAG GAG AAA TAT CAG C
3'end primer (SEQ ID NO: 18)
CGC TGA TCA TTA TAG CAA AAT CCT TTC CAA GCC C
were designed to amplify the entire HIV-1 gp160 coding sequence (env open
reading frame) (Ratner et al., Nature 313, 277-284) by polymerase chain
reaction (PCR). In addition, a BcL1 enzyme recognition sequence was
included in each of the primers to facilitate cloning of the coding
sequence into a vector plasmid. After the gp160 gene was cloned and
inserted into a baculovirus transfer vector pvl 1393, by techniques well
known in the art, the co-transfection was performed using the procedure
provided by the Baculo-Gold System (Pharmigen). The recombinant virus was
isolated to express a fully glycosylated gp160 polyprotein, the precursor
of HIV-1 env gene products.
EXAMPLE 3
Construction of Recombinant Baculovirus to Express HIV-1 pol 97
The recombinant virus expressing pol 97 was constructed as disclosed in Hu
et al., 1991, Proc. Natl. Acad. Sci., 88, 4596-4600.
EXAMPLE 4
SF9 Cell Culture and HIV Recombinant Protein Production
Spodoptera frugiperda (Sf9) cells are grown in monolayer (175 cm.sup.2
Falcon tissue culture flasks) or roller bottle (850 cm.sup.2 cell culture
bottles) cultures. The medium used for either culture condition is Gibco
BRL Sf900 medium supplemented with 100 U/ml each of sodium penicillin G
and streptomycin sulphate (Gibco BRL) or Sigma TNM-FH medium supplemented
with 10% (v/v) fetal bovine serum (Gibco BRL) and 100 U/ml each of the
above antibiotics.
The cells are infected at an moi of 5-10 with recombinant baculovirus
(Autographa californica). Harvest of the intracellular protein (protein
contained within the cells prior to cell lysis) was done using the same
procedure for both the monolayer and roller bottle cultures after an
optimum time of 72 hours post infection. The cells were released from the
surface of the flask or bottle and the suspension was spun down for 10
minutes at 250.times.g at ambient temperature on a Beckman GP centrifuge.
The cell pellet and the supernatant were then further processed. The
supernatant (extracelluar protein) was transferred to Beckman Ultraclear
tubes and spun down on a Beckman L8-80M ultracentrifuge at 26,000 rpm
(SW28 rotor) at 20.degree. C. for 1.5 hours. The pellet was resuspended in
1-2mL of phosphate buffered saline (PBS, 2.67 mM KCl, 1.15 mM KH.sub.2
PO.sub.4 137.9 mM NaCl, 8.06 mM Na.sub.2 HPO.sub.4 pH 7.4) and then stored
at -70.degree. C. until use.
The cell pellet was washed in 30 mL of PBS and spun down again in the GP
centrifuge. The pellet was resuspended in 10 mL of PBS then sonicated on
ice for 45-50 seconds at 40% power (Cole-Parmer Ultrasonic Homogenizer
4710). The cell lysates were spun down on the GP centrifuge at 900.times.g
for 10 minutes. The pellet fraction I, was resuspended in 1-2 mL of PBS
and was held at 4.degree. C. until use. The supernatant was spun down in a
Sorvall RC-5 centrifuge with an SS-34 rotor at 10,000 rpm at 4.degree. C.
for 30 minutes. The pellet fraction II, was resuspended in 1-2 mL of PBS
and held at 4.degree. until use. The supernatant was spun down on the
Beckmann ultracentrifuge under the same conditions as the extracelluar
protein. The resulting pellet, fraction III, was resuspended in 1-2 mL of
PBS and the supernatant was discarded. Fractions II and III were combined
to form the intracellular protein stock. The resulting pellet was further
purified by sucrose gradient (20%-60%) ultracentrifugation at 26000K rpm
(SW28 rotor) for 3 hours at 20.degree. C. The purified insoluble proteins
were washed in PBS and then spun down on an IEC Micro-MB centrifuge for 5
minutes at room temperature. The pellet was washed twice more then stored
at -70.degree. C. until further use.
EXAMPLE 5
Flow Cytometry--r-FIFA Assay for the Detection of HIV-1 Antibodies
1. Flow Cytometry
Flow cytometric analysis was performed on a Becton Dickinson FACSort
equipped with an argon ion laser tuned at 488 nm. Data acquisition was
done with Lysus II 2.0 software, version 1.1 (Becton Dickinson). Forward
light scattering, orthogonal light scattering and two fluorescent signals
were determined on logarithmic settings for each of 20,000 events and
stored in data files. Detector settings had been determined and stored in
data files for recall by the operator. Data analysis was also performed
with the Lysis 2.0 software. A two dimensional dot intensity plot of
forward light scatter versus orthogonal light scatter was observed on
ungated events. A region (R1) was set on the dot plot and single parameter
histograms of FL1 (green emission for FITC is 530 nm) and FL2 (red
emission for R-PE is 585 nm) were examined. The median fluorescence
channel was used to determine positivity of the test samples. This
analysis procedure has also been automated and stored as a command file.
2. r-FIFA Procedure
In 1.5 mL microcentrifuge tubes (Sarstedt) 4 .mu.L of control or sample
(plasma or serum) and a pre-determined amount of recombinant protein were
combined along with PBS (containing NaN.sub.3) to a final volume of 100
.mu.L. Most of the tests were done with a mixture of gag-p45 and
gag-gp41-B recombinant proteins. The rest were done with a single antigen
as indicated. The tubes were gently vortexed then incubated at ambient
temperature for 20 minutes on a rocker. The mixture was washed in 1 mL of
PBS/tube and spun down for 5 minutes, ambient temperature at 12
700.times.g on an IEC Micro-MB centrifuge (fixed speed). The supernatant
was aspirated and the pellet was gently resuspended in 1mL PBS and spun
down as before. The supernatant was aspirated and 10 .mu.L of FITC and/or
R-PE labelled antibody is added. The mixture was incubated for 20 minutes
at ambient temperature in the dark then washed two times with 1 ml/tube of
PBS as before. After the second aspiration, 500 .mu.L of PBS was added to
each sample tube and the mixture was sonicated for 10 seconds at the 40%
power setting with a 2 mm diameter probe attachment. The contents were
then transferred to 12.times.75 mm polystyrene tubes (Becton-Dickinson,
specific for the flow cytometer) and stored for 2 hours in the dark at
ambient temperature then read on the flow cytometer.
To determine the cut-off values of fluorescence intensity for r-FIFA for
each of the antigens (gag-p45, gag-gp41-C, pol97, and the mixture of
gag-p45 and gag-gp41-B proteins) 100 normal donors were tested by using
double staining with IgG FITC and IgM R-PE. Each sample was tested in
duplicate and reported in relation to the mean value of a known
seronegative control (S/N). The cut-off value was calculated by taking the
mean S/N value of the population (x) and adding two standard deviations (2
SD) as described by Sligh (Sligh, 1989, Amer. J. Clin. Pathol. 91,
210-214). For example, the cut-off value of fluorescence intensity of
gag-p45 was calculated as follows:
##EQU1##
Sample to cut-off (s/c) ratios of 1.0 or greater are considered positive.
EXAMPLE 6
Sensitivity and Reproducibility of r-FIFA
In order to determine the optimal serum or plasma dilution required for the
sensitivity test of r-FIFA, an HIV-1 seropositive plasma and a control
(negative plasma) were treated with gag-p45 as the antigen. FIG. 8 shows
the fluorescence histograms of the samples at different dilutions. The
fluorescence intensity of the positive sample sharply decreased as the
dilution increased. It was positive at 1:6400 (s/c=1.25). The 1:25
dilution was shown to be the optimal sample dilution for r-FIFA and this
dilution was used for further experiments.
Table 1 summarizes the results of r-FIFA for detection and analysis of
eight BBI anti-HIV-1 seroconversion panels and compares the sensitivity of
r-FIFA with currently licensed tests. Anti-HIV-1 antibodies were detected
by r-FIFA in the first bleed of seven BBI panels. The average increase in
sensitivity was greater than 20 days. FIG. 9 illustrates the fluorescence
histograms of samples in Panel D. The HIV-1 antibody (IgM) had become
positive three months before antibodies were detected by EIA. The antibody
titer dropped in sample D3 possibly because of HIV antigen-antibody
complex formation.
To evaluate the reproducibility of r-FIFA, two antigens gag-p45 and
gag-gp41-C were used for the analysis of panel D. The results of the two
assays were almost identical. The duplicates of each assay were highly
reproducible as shown by the overlapping of two line peaks which represent
the fluorescent intensity of each sample. In addition to the BBI panels,
three HIV-1 seroconversion panels from PHL were evaluated in a blind test
to confirm the sensitivity of r-FIFA (FIG. 10). The first bleed of all
three patients was seronegative in EIA and WB tests. However, IgG and IgM
HIV-1 antibodies were detected by r-FIFA. The antibody response pattern of
the three panels suggests that the patients were infected recently since
IgM was the dominant antibody. The first bleed from the patient PHL-A (who
donated blood for a transfusion) was negative by EIA in 1988. The
recipient was infected by the seronegative but HIV-1 infected blood. The
donor was identified as HIV-1 positive by testing in 1993 with a recently
licensed test which used conjugated second antibody to both human IgG and
IgM. The 1988 sample was still negative by this test but the 1993 sample
was strongly positive. In r-FIFA, both the 1988 and the 1993 samples were
positive for IgG and IgM. Furthermore, to compare the sensitivity of
r-FIFA with that of FDA licensed confirmatory tests including WB, and
radioimmunoprecipitation (RIPA), the BBI anti-HIV-1 1 ow titer panel was
tested with r-FIFA using each of the antigens gag-p45, gag-gp41 chimeric
proteins and pol97. The results are summarized in Table 2 and show that
r-FIFA is more sensitive than any of the FDA licensed confirmatory tests.
The antibodies to gag and env or to all three of these antigens were
positive by r-FIFA in 12 of the 14 samples. Only 8 to 10 were positive in
the four confirmatory tests. The results show that the pol97 is an
excellent antigen for HIV-1 antibody detection. Antibodies to pol97 were
detectable in 8 out of the 14 samples in r-FIFA. However, the antibodies
to pol gene products (p68, p51 and p31) were poorly detected in the FDA
licensed confirmatory tests. Only 1 to 4 of the 14 samples were found to
have antibodies to pol proteins on WB and RIPA. It has been reported that
antibodies to gag-encoded proteins appear first, followed closely by those
antibodies to env glycoproteins, then pol reactivity to p66 appear on WB.
Our studies show that the antibodies to pol97 were detected as early as
those to gag-p45 and gag-gp41 chimeric proteins as shown in FIG. 15. This
provides evidence to the effect that antibodies to pol gene products are
one of the important serological markers for early diagnosis of HIV-1
infection and monitoring of HIV-1 infection disease progression.
EXAMPLE 7
Specificity of r-FIFA
295 random donor samples, 105 EIA/WB positive samples and 128 EIA repeat
reactive or positive/WB indeterminate samples were tested by r-FIFA. Only
1 of the 295 random donor samples was positive. One of the positive
samples from the random donor was negative in EIA but was positive by
radioimmunoprecipitation (r-RIPA). All 105 positive samples were positive
by r-FIFA. Only 30 of 128 (23%) EIA repeat reactive WB indeterminate
samples were still positive. These results indicate that r-FIFA's
specificity is higher than that of EIA and WB.
EXAMPLE 8
Antibody Response During the Window Period
FIG. 11 demonstrates the patterns of HIV-1 antibody production in eight BBI
panels. A typical primary response was found in panels J and K. Panels D
and R show a similar pattern of antibody production. However, in panels E,
H, P and Q, the HIV-1 IgG antibody remained at a certain level for a long
period (over two or three months) prior to seroconversion as detected by
licensed tests. The IgM antibody became positive later at a low level or
at the same time (panel Q) as did IgG antibodies. The pattern of E, H, P
and Q is apparently different from the pattern of D, J, K and R.
Presumably these individuals were not recently infected by HIV-1. For some
reason, virus-antigen concentration rose, stimulating the antibody
response. The anti-HIV-1 IgG antibodies jumped up to a high level in a
shorter time (less than a week as seen in Q and R) indicating a possible
secondary immune response. FIG. 12 shows the fluorescence histograms of
the samples in panel K, indicating the kinetics of the specific IgG, IgM
and IgA production during the window period. The IgM antibodies were
detected first before the antigen was detected, and then IgA and IgG
antibodies appeared about two weeks after the antigen level peaked. The
IgM antibody response to HIV-1 proteins overlapped with the HIV-1 antigen
peak and started before the antigen was detected.
EXAMPLE 9
Differences in Antibody Response to Different Antigens gag-p45, gag-gp41-C
and pol97 in r-FIFA
Chimeric gag-gp41-C binds more IgG antibodies than gag-p45 and detects them
earlier. Little difference was found in detection of HIV-1 IgM antibodies
(FIGS. 13 and 14). HIV-1 IgG antibodies to pol97 were detected as early as
gag-gp41-C (FIG. 15). IgM antibody to pol97 was very low during this
"window" period.
EXAMPLE 10
Comparison of r-FIFA Assay with gag-p45, gag-gp 41-C, pol97, gp 160 and
Mixture of gag-45, pol97 and gp 160
In a preferred embodiment, a mixture of equal amounts of gag-p45, pol97 and
gp 160 is used in the r-FIFA. The proteins were prepared as described in
the previous examples and the r-FIFA was conducted as previously
described. An anti-HIV-1 low titer performance panel (PRB 104) was
utilized. The results are reported in Table 3. The testing with a mixture
of all three proteins was carried out with 5 samples. The results with the
mixture were positive in all instances. This shows the sensitivity of the
procedure with a mixture of the proteins. In instances where the results
were negative for each of the four proteins individually, the results with
the mixture were negative as well (sample 10) and where the results were
positive with each of the proteins individually, the results of the
mixture were positive as well (sample 7). The use of a mixture of the
proteins is advantageous in a situation where the results with each of the
proteins all not all positive or negative (samples 3, 11 and 14). In all
instances where some of the individual results were negative, the use of a
mixture of proteins allowed a final determination and gave positive
results. The gag, gp and pol polyproteins cover over 90% of the viral
structural proteins and allow r-FIFA to be used as a confirmatory test
because of its high sensitivity, specificity and its ability to identify
antibodies directed to individual HIV-1 polyproteins.
EXAMPLE 11
Flow Cytometry--r-FIFA Assay for the Detection of Hepatitis B Antibodies
The Hepatitis B core antigen was prepared as is known in the art and as is
described for example in Takehara et al., 1988, J. Gen. Virology,
69:2763-2777. Recombinant baculoviruses were constructed to express the
hepatitis B core antigen in the same manner as described in Example 1 in
relation to HIV-1 gag p45 and chimeric gag-gp 41 proteins. The r-FIFA
assay for the detection of antibodies to Hepatitis B core antigens was
conducted as set out in Example 5 in relation to the detection of HIV-1
antibodies. The results of a comparison of sensitivity between licensed
EIA and RIA tests for Hepatitis B and r-FIFA using standard BBI panels are
set out in Table 4.
The above examples demonstrate that r-FIFA is more sensitive than licensed
screening and confirmatory tests for detection of early antibodies to
HIV-1. At least one class of anti-HIV-1 specific IgG, IgM or IgA was
detected during the window period of HIV-1 infected individuals. We have
created a unique way to detect early antibodies using insoluble forms of
recombinant proteins as autologous carriers through a flow cytometer. Two
patterns of antibody response were observed, which may represent two types
of immune response: primary and secondary.
On average for the tests which were in use in 1990 the window period was 45
days. Since then the tests have increased sensitivity for HIV antibody
detection resulting in earlier detection of seroconversion and a
significant decrease of approximately 12-13 days in the length of the
infectious window period (Dodd, 1990, Arch. Pathol. Lab. Med. 114,
240-245). An analysis of the records made in 19 American National Red
Cross regions in 1992 and 1993 showed that the window period estimated
using third generation tests was 25 days on average Petersen et al., 1994,
Transfusion 34,283-289). As shown in Table 1, the average increase in
sensitivity using the method of the present invention was greater than 20
days. The r-FIFA of the present invention is a novel serological test,
which uses insoluble polyproteins. The insoluble antigens are easily
processed and purified without denaturation, keeping intact their natural
molecular folding which may optimize the presentation of antigen epitopes.
This may be one of the explanations to account for r-FIFA's higher
sensitivity in detection of HIV-1 early antibodies. Furthermore, an
agglutination effect involving the reaction between IgM antibodies and the
antigens in r-FIFA may enhance the sensitivity for detection of specific
antibodies because all the insoluble polyproteins used are particulate
antigens. This is demonstrated in FIG. 16. In general, IgM antibodies are
more efficient in agglutination than IgG antibodies because of their size
as well as their additional binding sites.
The assay of the present invention (r-FIFA) is much more sensitive than any
of the currently licensed tests used for analysis of BBI seroconversion
panels to detect very early HIV-1 antibodies during the "window" period.
Both anti-HIV-1 specific IgG and IgM have been found prior to the
seroconversion of seroconversion panels. Some of them contain primarily
anti-HIV-1 IgG or IgM. Some individuals have both anti-HIV-1 IgG and IgM.
This appears to depend on the time of primary infection, appearance of
antigenemia and the situation of the immune system of the HIV-1 infected
individuals. Detection, quantification and differentiation of anti-HIV-1
antibodies in the "window" period have permitted a further understanding
of the immune response and HIV-1 immunopathogenesis in this "immunological
silent" period. The development of r-FIFA has allowed the use of insoluble
proteins as antigens to detect antibodies. These insoluble proteins can be
easily produced on a large scale using a baculovirus expression system.
While the r-FIFA procedure for use in the detection of HIV-1 infection is
described in relation to gag p45, gag-gp 41, gp 160 and pol97 recombinant
proteins, it will be understood that a mixture of these proteins as
exemplified in Example 10 can be used as well as any number of other
proteins and need not be recombinant proteins prepared in the baculovirus
expression system. The proteins are not limited to those specifically
exemplified. While the examples and the results set out above pertain to
the use of the recombinant proteins in the detection and treatment of
HIV-1, it is understood that the examples are not meant to be limiting.
The invention would have applicability to the detection and treatment of
HIV-2 using an insoluble form of a protein equivalent to gag p45 in HIV-1
as well as have applicability in the detection and treatment of other
viral infections. This is illustrated in Example 11 where r-FIFA is used
in the detection of hepatitis B. The invention also has applicability in
the detection of other viral infections such as hepatitis C, HTLV-1/2
(human T-cell leukemia) or HTLV-II and has applicability for any
virus-mediated condition where the virus proteins exist in insoluble form.
Indeed, it is not limited to the detection of viral infections but has
application for the detection of any disease state where an insoluble form
of an antigen can be used in the assay of the present invention for the
detection of antibodies. It is also contemplated that chimeric proteins
can be constructed comprising several antigenic domains so that one test
could be used for the detection of several disease states, for example,
HIV-1/2, HTLV-1/2, hepatitis B and hepatitis C.
Furthermore, it is also understood that where recombinant proteins are used
in the assay of the present invention, any expression system sharing the
advantages of the baculovirus expression system could also be used.
All publications referred to in the disclosure are hereby incorporated by
reference.
While the present invention has been described in connection with a
specific embodiment thereof and in a specific use, various modifications
will occur to those skilled in the art without departing from the spirit
and scope of the invention as set forth in the appended claims. I
therefore wish to embody within the scope of the patent which may be
granted hereon all such embodiments as reasonably and properly come within
the scope of my contribution to the art.
TABLE 1
__________________________________________________________________________
Comparison of the Sensitivity Between r-FIFA and Licensed Tests for
HIV-1 Antibodies in Anti-HIV-1 Seroconversion Panels (BBI)
Time
# of Span Members
Antibody Reactivity of
Each Panel Member
Code
Members
(Days)
Antigen +
1 2 3 4 5 6 7 8 9 10 Tests
__________________________________________________________________________
D 5 101 0 - - - + + EIA
+ + (+) + + r-FIFA
E 10 126 2 - - - - - - - - - + EIA
+ + + (+) - + (+) + + + r-FIFA
H 6 28 0 - - - - - + EIA
+ + + + (+) + r-FIFA
J 7 40 0 - - + + + + + EIA
+ + + + + + + r-FIFA
K 10 36 0 - - - (+) (+) (+) (+) + + + EIA
+ + + + + + + + + + r-FIFA
P 6 35 1 - - - - + + EIA
+ + (+) + + + r-FIFA
Q 7 72 5 - - - - (+) (+) + EIA
+ + - (+) + + + r-FIFA
R 6 21 2 - (+) (+) + + + EIA
- (+) + + + + r-FIFA
__________________________________________________________________________
EIA data shown in this table is a summary of 13 licensed test results fro
BBI. Both antiHIV-specific IgG and IgM were detected by rFIFA using
fluorescence conjugated second antibodies to human IgG and IgM. The
antiHIV-1 antibodies IgG and IgM were detected separately by double
staining with IgG FITC and IgM RPE. If the s/c value .gtoreq. 0.9 but <
1.0 it is considered to be weak reactive (+); the s/c value of 1.0 or
greater is considered positive +. The weak reactivity # (+) by EIA means
that the sample is positive in some tests and negative in others.
TABLE 2
__________________________________________________________________________
Comparison between r-FIFA and FDA licensed confirmatory tests based on
sensitivity for detection of antibodies to HIV-1 proteins encoded in the
three open reading frames of HIV-1 genome in the anti-HIV-1 low titer
panel PRB104
FDA Licensed Tests
PRB104 I.D.
BioRad WB* Ortho/Cambridge WB
Organon Teknika WB
HIV-1 RIPA RL15
HIV-1 r-FIFA
Number gag
pol
env
Res.
gag
pol
env
Res.
gag
pol
env
Res.
gag
pol
env
Res.
gag
pol
env.sup..dagger.
Res.
__________________________________________________________________________
01 + - + + + + + + + - + + + - + + + + + +
02 + - + + + - + + + - + + + - + + + - + +
03 + - + + + - + + + + + + + + + + + - + +
04 + - + + + - + + + - + + + - + + + + + +
05 + - + + + + + + + + + + + + + + + + + +
06 + + + + + - + + + + + + + + + + + + + +
07 + - - IND + - - IND + - - IND - - + IND + + + +
08 - - - - + - - IND + - - IND - - + IND + + + +
09 + - + + + - + + + - + + - - + IND + + + +
10.sup..dagger-dbl. - - - - - - - - - - - - - - - - - - - -
11 + - + + + - + + + - + + + - + + + - + +
12 + - + + + - + + + - + + + + + + + - + +
13 + - + + + - - IND + - f160 + - - + IND + + + +
14 f24 - - IND - - - - f24 - - IND - - + IND + - - IND
15 - - - - - - - - - - - - - - - - - - - -
TOTAL 12 1 10 10 12 2 9 9 13 3 10 10 8 4 13 8 13 8 12 12
POSITIVES
gag + env + =
71% 64% 71% 57% 86%
gag + pol + 7% 14% 21% 29% 57%
env + =
__________________________________________________________________________
*Western Blots were interpreted using CDC/ASTPHLD criteria. All Western
Blots were performed at Boston Biomedica.
.sup..dagger. The chimeric gaggp41(C, B) was used for detection of
antibodies to env. Later, the results were confirmed using insoluble gp16
polyprotein as antigen in rFIFA.
.sup..dagger-dbl. PRB104 10 is the negative control.
f = faint, + = positive, - = negative, IND = indeterminate, RES = result
TABLE 3
______________________________________
Detection of Antibodies to HIV-1 in Samples of Anti-HIV-1 Low Titer
Performance Pabels (PRB104) using Different Antigens
ANTIGENS
mixture of
gag-p45,
PRB104 I.D. gag-p45 gag-gp41-C pol97 gp160 pol97 and
NUMBER s/c s/c s/c s/c gp160 s/c
______________________________________
01 1.64 1.18 1.10 1.17 ND
02 1.35 1.20 0.80 1.24 ND
03 1.11 1.29 0.87 1.00 2.30
04 1.31 1.31 1.10 1.37 ND
05 1.49 1.08 1.47 1.03 ND
06 2.20 1.22 1.70 1.23 ND
07 1.27 1.00 1.80 1.00 1.36
08 1.36 1.06 1.24 1.22 ND
09 1.34 1.00 1.32 1.00 ND
10 0.89 0.63 0.61 0.72 0.71
11 1.45 1.04 0.80 1.00 1.46
12 1.38 1.06 0.79 1.16 ND
13 1.59 0.93 1.47 1.22 ND
14 1.09 0.78 0.58 0.78 1.16
15 0.81 0.77 0.86 0.54 ND
______________________________________
TABLE 4
__________________________________________________________________________
Comparison of Sensitivity Between Licensed Tests and r-FIFA in
Detection of Antibodies to HBc
HBsAg
ANTI-HBs
ANTI-HBc ANTI-HBc ANTI-HBc ANTI-HBc ABBOTT ABBOTT
IgM ABBOTT IgM ABBOTT IgM ABBOTT IgM ABBOTT ANTI-HBc RIA EIA ANTI-HBc
MEMBER EIA PROC A EIA PROC B RIA PROC A RIA PROC B IgM SORIN PROC A
PROC B r-FIFA
I.D. MA- (S/CO) (S/CO) (S/CO) (S/CO) EIA (S/CO)
(S/CO)
IgG
IgM
NUMBER
TRIX
BBI BBI RL1 BBI BBI RL1 BBI BBI BBI (s/c)
(s/c)
__________________________________________________________________________
PHE201-01
P 4.5 4.8 4.7 5.8 5.3 9.2 8.2 114 0.2 5.6
2.7
PHE201-02 P 4.0 3.9 4.6 4.8 5.1 6.0 9.9 99 0.2 8.7 2.71
PHE201-03 P 13.0 5.5 4.6 11.5 12.7 13.1 12.0 100 0.2 7.33 6.09
PHE201-04 P 0.4 0.5 0.6 0.5 0.5 0.6 2.2 131 0.4 0.58 0.52
PHE201-05 P 6.5 5.1 4.6 5.0 6.0 8.2 10.1 5 0.2 6.93 4.66
PHE201-06 P 3.0 3.0 4.1 3.3 2.7 4.2 7.9 106 0.2 5.55 2.11
PHE201-07 P 2.7 2.4 3.7 3.7 4.0 4.3 8.6 26 0.1 6.83 2.46
PHE201-08 P 3.7 3.3 4.6 3.5 4.3 5.9 10.5 117 0.2 7.60 4.19
PHE201-09 S 3.7 3.5 4.3 3.7 3.5 5.0 9.6 129 0.1 7.08 2.84
PHE201-10 P 1.5 1.8 3.5 1.6 1.9 4.5 5.3 98 0.2 3.87 2.72
PHE201-11 P 0.4 0.5 0.8 0.4 0.4 0.5 1.8 94 0.6 3.72 0.81
PHE201-12 P 7.3 5.5 4.7 6.7 7.8 9.1 10.1 15 0.2 3.62 4.71
PHE201-13 P 5.5 4.4 4.7 6.8 5.2 7.3 10.4 88 0.2 8.60 4.71
PHE201-14 P 5.8 5.1 4.7 5.6 6.3 8.0 10.6 96 0.2 4.44 3.42
PHE201-15 S 8.5 8.1 4.7 9.0 10.0 10.8 11.2 113 0.1 9.25 5.94
PHE201-16 P 9.1 5.5 4.7 8.3 8.4 10.8 11.0 103 0.2 9.77 5.16
PHE201-17 P 5.1 5.0 4.7 7.0 7.1 8.9 10.9 103 0.2 8.55 3.64
PHE201-18 S 1.8 1.9 2.6 1.7 2.0 2.6 6.8 80 0.1 6.65 2.6
PHE201-19 S 3.9 3.0 4.1 3.0 3.0 4.8 8.5 108 0.2 5.04 3.54
PHE201-20 S 2.2 2.4 2.6 2.2 2.7 2.1 6.3 117 0.2 7.88 2.59
PHE201-21 P 3.1 2.8 3.3 2.7 2.5 3.7 8.2 128 0.1 5.41 2.11
PHE201-22 P 2.5 3.1 3.1 2.6 2.4 3.7 8.9 101 0.2 1.54 1.17
PHE201-23 P 2.1 2.3 2.8 2.8 2.7 2.6 6.1 152 0.2 6.55 1.89
PHE201-24 P 3.8 2.8 3.8 2.5 3.4 4.0 8.9 113 0.3 2.69 1.87
PHE201-25 P 2.7 3.1 4.2 2.9 3.3 4.3 10.3 114 0.2 9.13 4.89
__________________________________________________________________________
All panel members have been found positive by a test for HBsAg and
negative by a test for antiHIV-1.
EIA and RIA results were generated using commercially available FDA
approved antiHBc IgM screening tests, performed at BBI and at a nationall
recognized noncommercial referee laboratory (RL1) by individuals who
routinely use these procedures. All numeric results are means of
duplicates, expressed as specimen absorbance to cutoff ratios (s/co).
Ratios .gtoreq. 1.0 are considered reactive.
Specimens are undiluted aliquots form serum (S) or plasma (P) units
collected from asymptomatic blood donors in 1989 and 1990.
__________________________________________________________________________
# SEQUENCE LISTING
- - - - (1) GENERAL INFORMATION:
- - (iii) NUMBER OF SEQUENCES: 21
- - - - (2) INFORMATION FOR SEQ ID NO:1:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1314 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
- - ATGGGTGCGA GAGCGTCAGT ATTAAGCGGG GGAGAATTAG ATCGATGGGA AA -
#AAATTCGG 60
- - TTAAGGCCAG GGGGAAAGAA AAAATATAAA TTAAAACATA TAGTATGGGC AA -
#GCAGGGAG 120
- - CTAGAACGAT TCGCAGTTAA TCCTGGCCTG TTAGAAACAT CAGAAGGCTG TA -
#GACAAATA 180
- - CTGGGACAGC TACAACCATC CCTTCAGACA GGATCAGAAG AACTTAGATC AT -
#TATATAAT 240
- - ACAGTAGCAA CCCTCTATTG TGTGCATCAA AGGATAGAGA TAAAAGACAC CA -
#AGGAAGCT 300
- - TTAGACAAGA TAGAGGAAGA GCAAAACAAA AGTAAGAAAA AAGCACAGCA AG -
#CAGCAGCT 360
- - GACACAGGAC ACAGCAGTCA GGTCAGCCAA AATTACCCTA TAGTGCAGAA CA -
#TCCAGGGG 420
- - CAAATGGTAC ATCAGGCCAT ATCACCTAGA ACTTTAAATG CATGGGTAAA AG -
#TAGTAGAA 480
- - GAGAAGGCTT TCAGCCCAGA AGTAATACCC ATGTTTTCAG CATTATCAGA AG -
#GAGCCACC 540
- - CCACAAGATT TAAACACCAT GCTAAACACA GTGGGGGGAC ATCAAGCAGC CA -
#TGCAAATG 600
- - TTAAAAGAGA CCATCAATGA GGAAGCTGCA GAATGGGATA GAGTACATCC AG -
#TGCATGCA 660
- - GGGCCTATTG CACCAGGCCA GATGAGAGAA CCAAGGGGAA GTGACATAGC AG -
#GAACTACT 720
- - AGTACCCTTC AGGAACAAAT AGGATGGATG ACAAATAATC CACCTATCCC AG -
#TAGGAGAA 780
- - ATTTATAAAA GATGGATAAT CCTGGGATTA AATAAAATAG TAAGAATGTA TA -
#GCCCTACC 840
- - AGCATTCTGG ACATAAGACA AGGACCAAAA GAACCTTTTA GAGACTATGT AG -
#ACCGGTTC 900
- - TATAAAACTC TAAGAGCCGA GCAAGCTTCA CAGGAGGTAA AAAATTGGAT GA -
#CAGAAACC 960
- - TTGTTGGTCC AAAATGCGAA CCCAGATTGT AAGACTATTT TAAAAGCATT GG -
#GACCAGCG 1020
- - GCTACACTAG AAGAAATGAT GACAGCATGT CAGGGAGTAG GAGGACCCGG CC -
#ATAAGGCA 1080
- - AGAGTTTTGG CTGAAGCAAT GAGCCAAGTA ACAAATACAG CTACCATAAT GA -
#TGCAGAGA 1140
- - GGCAATTTTA GGAACCAAAG AAAGATGGTT AAGTGTTTCA ATTGTGGCAA AG -
#AAGGGCAC 1200
- - ACAGCCAGAA ATTGCAGGGC CCCTAGGAAA AAGGGCTGTT GGAAATGTGG AA -
#AGGAAGGA 1260
- - CACCAAATGA AAGATTGTAC TGAGAGACAG GCTAATTTTT TAGGGAAGAT CT - #AA
1314
- - - - (2) INFORMATION FOR SEQ ID NO:2:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 437 amino - #acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: peptide
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
- - Met Gly Ala Arg Ala Ser Val Leu Ser Gly Gl - #y Glu Leu Asp Arg Trp
1 5 - # 10 - # 15
- - Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Ly - #s Lys Tyr Lys Leu Lys
20 - # 25 - # 30
- - His Ile Val Trp Ala Ser Arg Glu Leu Glu Ar - #g Phe Ala Val Asn Pro
35 - # 40 - # 45
- - Gly Leu Leu Glu Thr Ser Glu Gly Cys Arg Gl - #n Ile Leu Gly Gln Leu
50 - # 55 - # 60
- - Gln Pro Ser Leu Gln Thr Gly Ser Glu Glu Le - #u Arg Ser Leu Tyr Asn
65 - #70 - #75 - #80
- - Thr Val Ala Thr Leu Tyr Cys Val His Gln Ar - #g Ile Glu Ile Lys Asp
85 - # 90 - # 95
- - Thr Lys Glu Ala Leu Asp Lys Ile Glu Glu Gl - #u Gln Asn Lys Ser Lys
100 - # 105 - # 110
- - Lys Lys Ala Gln Gln Ala Ala Ala Asp Thr Gl - #y His Ser Asn Gln Val
115 - # 120 - # 125
- - Ser Gln Asn Tyr Pro Ile Val Gln Asn Ile Gl - #n Gly Gln Met Val His
130 - # 135 - # 140
- - Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Tr - #p Val Lys Val Val Glu
145 1 - #50 1 - #55 1 -
#60
- - Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Me - #t Phe Ser Ala Leu
Ser
165 - # 170 - # 175
- - Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Me - #t Leu Asn Thr Val Gly
180 - # 185 - # 190
- - Gly His Gln Ala Ala Met Gln Met Leu Lys Gl - #u Thr Ile Asn Glu Glu
195 - # 200 - # 205
- - Ala Ala Glu Trp Asp Arg Val His Pro Val Hi - #s Ala Gly Pro Ile Ala
210 - # 215 - # 220
- - Pro Gly Gln Met Arg Glu Pro Arg Gly Ser As - #p Ile Ala Gly Thr Thr
225 2 - #30 2 - #35 2 -
#40
- - Ser Thr Leu Gln Glu Gln Ile Gly Trp Met Th - #r Asn Asn Pro Pro
Ile
245 - # 250 - # 255
- - Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Il - #e Leu Gly Leu Asn Lys
260 - # 265 - # 270
- - Ile Val Arg Met Tyr Ser Pro Thr Ser Ile Le - #u Asp Ile Arg Gln Gly
275 - # 280 - # 285
- - Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Ar - #g Phe Tyr Lys Thr Leu
290 - # 295 - # 300
- - Arg Ala Glu Gln Ala Ser Gln Glu Val Lys As - #n Trp Met Thr Glu Thr
305 3 - #10 3 - #15 3 -
#20
- - Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Ly - #s Thr Ile Leu Lys
Ala
325 - # 330 - # 335
- - Leu Gly Pro Ala Ala Thr Leu Glu Glu Met Me - #t Thr Ala Cys Gln Gly
340 - # 345 - # 350
- - Val Gly Gly Pro Gly His Lys Ala Arg Val Le - #u Ala Glu Ala Met Ser
355 - # 360 - # 365
- - Gln Val Thr Asn Ser Ala Thr Ile Met Met Gl - #n Arg Gly Asn Phe Arg
370 - # 375 - # 380
- - Asn Gln Arg Lys Ile Val Lys Cys Phe Asn Cy - #s Gly Lys Glu Gly His
385 3 - #90 3 - #95 4 -
#00
- - Thr Ala Arg Asn Cys Arg Ala Pro Arg Lys Ly - #s Gly Cys Trp Lys
Cys
405 - # 410 - # 415
- - Gly Lys Glu Gly His Gln Met Lys Asp Cys Th - #r Glu Arg Gln Ala Asn
420 - # 425 - # 430
- - Phe Leu Gly Lys Ile
435
- - - - (2) INFORMATION FOR SEQ ID NO:3:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 354 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..354
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
- - GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GT - #G GTG CAG AGA GAA AAA
48
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys
1 5 - # 10 - # 15
- - AGA GCA GTG GGA ATA GGA GCT TTG TTC CTT GG - #G TTC TTG GGA GCA GCA
96
Arg Ala Val Gly Ile Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala Ala
20 - # 25 - # 30
- - GGA AGC ACT ATG GGC GCA GCG TCA ATG ACG CT - #G ACG GTA CAG GCC AGA
144
Gly Ser Thr Met Gly Ala Ala Ser Met Thr Le - #u Thr Val Gln Ala Arg
35 - # 40 - # 45
- - CAA TTA TTG TCT GGT ATA GTG CAG CAG CAG AA - #C AAT TTG CTG AGG GCT
192
Gln Leu Leu Ser Gly Ile Val Gln Gln Gln As - #n Asn Leu Leu Arg Ala
50 - # 55 - # 60
- - ATT GAG GCG CAA CAG CAT CTG TTG CAA CTC AC - #A GTC TGG GGC ATC AAG
240
Ile Glu Ala Gln Gln His Leu Leu Gln Leu Th - #r Val Trp Gly Ile Lys
65 - # 70 - # 75 - # 80
- - CAG CTC CAG GCA AGA ATC CTG GCT GTG GAA AG - #A TAC CTA AAG GAT CAA
288
Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Ar - #g Tyr Leu Lys Asp Gln
85 - # 90 - # 95
- - CAG CTC CTG GGG ATT TGG GGT TGC TCT GGA AA - #A CTC ATT TGC ACC ACT
336
Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Ly - #s Leu Ile Cys Thr Thr
100 - # 105 - # 110
- - GCT GTG CCT TGG AAT GCT - # - #
- # 354
Ala Val Pro Trp Asn Ala
115
- - - - (2) INFORMATION FOR SEQ ID NO:4:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 118 amino - #acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
- - Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys
1 5 - # 10 - # 15
- - Arg Ala Val Gly Ile Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala Ala
20 - # 25 - # 30
- - Gly Ser Thr Met Gly Ala Ala Ser Met Thr Le - #u Thr Val Gln Ala Arg
35 - # 40 - # 45
- - Gln Leu Leu Ser Gly Ile Val Gln Gln Gln As - #n Asn Leu Leu Arg Ala
50 - # 55 - # 60
- - Ile Glu Ala Gln Gln His Leu Leu Gln Leu Th - #r Val Trp Gly Ile Lys
65 - # 70 - # 75 - # 80
- - Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Ar - #g Tyr Leu Lys Asp Gln
85 - # 90 - # 95
- - Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Ly - #s Leu Ile Cys Thr Thr
100 - # 105 - # 110
- - Ala Val Pro Trp Asn Ala
115
- - - - (2) INFORMATION FOR SEQ ID NO:5:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 342 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..342
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
- - AGG GCT ATT GAG GCG CAA CAG CAT CTG TTG CA - #A CTC ACA GTC TGG GGC
48
Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gl - #n Leu Thr Val Trp Gly
1 5 - # 10 - # 15
- - ATC AAG CAG CTC CAG GCA AGA ATC CTG GCT GT - #G GAA AGA TAC CTA AAG
96
Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Va - #l Glu Arg Tyr Leu Lys
20 - # 25 - # 30
- - GAT CAA CAG CTC CTG GGG ATT TGG GGT TGC TC - #T GGA AAA CTC ATT TGC
144
Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Se - #r Gly Lys Leu Ile Cys
35 - # 40 - # 45
- - ACC ACT GCT GTG CCT TGG AAT GCT AGT TGG AG - #T AAT AAA TCT CTG GAA
192
Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Se - #r Asn Lys Ser Leu Glu
50 - # 55 - # 60
- - CAG ATT TGG AAT AAC ATG ACC TGG ATG GAG TG - #G GAC AGA GAA ATT AAC
240
Gln Ile Trp Asn Asn Met Thr Trp Met Glu Tr - #p Asp Arg Glu Ile Asn
65 - # 70 - # 75 - # 80
- - AAT TAC ACA AGC TTA ATA CAC TCC TTA ATT GA - #A GAA TCG CAA AAC CAG
288
Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Gl - #u Glu Ser Gln Asn Gln
85 - # 90 - # 95
- - CAA GAA AAG AAT GAA CAA GAA TTA TTG GAA TT - #A GAT AAA TGG GCA AGT
336
Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Le - #u Asp Lys Trp Ala Ser
100 - # 105 - # 110
- - TTG TGG - # - # -
# 342
Leu Trp
- - - - (2) INFORMATION FOR SEQ ID NO:6:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 114 amino - #acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
- - Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gl - #n Leu Thr Val Trp
Gly
1 5 - # 10 - # 15
- - Ile Lys Gln Leu Gln Ala Arg Ile Leu Ala Va - #l Glu Arg Tyr Leu Lys
20 - # 25 - # 30
- - Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Se - #r Gly Lys Leu Ile Cys
35 - # 40 - # 45
- - Thr Thr Ala Val Pro Trp Asn Ala Ser Trp Se - #r Asn Lys Ser Leu Glu
50 - # 55 - # 60
- - Gln Ile Trp Asn Asn Met Thr Trp Met Glu Tr - #p Asp Arg Glu Ile Asn
65 - # 70 - # 75 - # 80
- - Asn Tyr Thr Ser Leu Ile His Ser Leu Ile Gl - #u Glu Ser Gln Asn Gln
85 - # 90 - # 95
- - Gln Glu Lys Asn Glu Gln Glu Leu Leu Glu Le - #u Asp Lys Trp Ala Ser
100 - # 105 - # 110
- - Leu Trp
- - - - (2) INFORMATION FOR SEQ ID NO:7:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 528 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..528
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
- - GGA GTA GCA CCC ACC AAG GCA AAG AGA AGA GT - #G GTG CAG AGA GAA AAA
48
Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys
1 5 - # 10 - # 15
- - AGA GCA GTG GGA ATA GGA GCT TTG TTC CTT GG - #G TTC TTG GGA GCA GCA
96
Arg Ala Val Gly Ile Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala Ala
20 - # 25 - # 30
- - GGA AGC ACT ATG GGC GCA GCG TCA ATG ACG CT - #G ACG GTA CAG GCC AGA
144
Gly Ser Thr Met Gly Ala Ala Ser Met Thr Le - #u Thr Val Gln Ala Arg
35 - # 40 - # 45
- - CAA TTA TTG TCT GGT ATA GTG CAG CAG CAG AA - #C AAT TTG CTG AGG GCT
192
Gln Leu Leu Ser Gly Ile Val Gln Gln Gln As - #n Asn Leu Leu Arg Ala
50 - # 55 - # 60
- - ATT GAG GCG CAA CAG CAT CTG TTG CAA CTC AC - #A GTC TGG GGC ATC AAG
240
Ile Glu Ala Gln Gln His Leu Leu Gln Leu Th - #r Val Trp Gly Ile Lys
65 - # 70 - # 75 - # 80
- - CAG CTC CAG GCA AGA ATC CTG GCT GTG GAA AG - #A TAC CTA AAG GAT CAA
288
Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Ar - #g Tyr Leu Lys Asp Gln
85 - # 90 - # 95
- - CAG CTC CTG GGG ATT TGG GGT TGC TCT GGA AA - #A CTC ATT TGC ACC ACT
336
Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Ly - #s Leu Ile Cys Thr Thr
100 - # 105 - # 110
- - GCT GTG CCT TGG AAT GCT AGT TGG AGT AAT AA - #A TCT CTG GAA CAG ATT
384
Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Ly - #s Ser Leu Glu Gln Ile
115 - # 120 - # 125
- - TGG AAT AAC ATG ACC TGG ATG GAG TGG GAC AG - #A GAA ATT AAC AAT TAC
432
Trp Asn Asn Met Thr Trp Met Glu Trp Asp Ar - #g Glu Ile Asn Asn Tyr
130 - # 135 - # 140
- - ACA AGC TTA ATA CAC TCC TTA ATT GAA GAA TC - #G CAA AAC CAG CAA GAA
480
Thr Ser Leu Ile His Ser Leu Ile Glu Glu Se - #r Gln Asn Gln Gln Glu
145 1 - #50 1 - #55 1 -
#60
- - AAG AAT GAA CAA GAA TTA TTG GAA TTA GAT AA - #A TGG GCA AGT TTG
TGG 528
Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Ly - #s Trp Ala Ser Leu Trp
165 - # 170 - # 175
- - - - (2) INFORMATION FOR SEQ ID NO:8:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 176 amino - #acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: protein
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
- - Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Va - #l Val Gln Arg Glu Lys
1 5 - # 10 - # 15
- - Arg Ala Val Gly Ile Gly Ala Leu Phe Leu Gl - #y Phe Leu Gly Ala Ala
20 - # 25 - # 30
- - Gly Ser Thr Met Gly Ala Ala Ser Met Thr Le - #u Thr Val Gln Ala Arg
35 - # 40 - # 45
- - Gln Leu Leu Ser Gly Ile Val Gln Gln Gln As - #n Asn Leu Leu Arg Ala
50 - # 55 - # 60
- - Ile Glu Ala Gln Gln His Leu Leu Gln Leu Th - #r Val Trp Gly Ile Lys
65 - # 70 - # 75 - # 80
- - Gln Leu Gln Ala Arg Ile Leu Ala Val Glu Ar - #g Tyr Leu Lys Asp Gln
85 - # 90 - # 95
- - Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Ly - #s Leu Ile Cys Thr Thr
100 - # 105 - # 110
- - Ala Val Pro Trp Asn Ala Ser Trp Ser Asn Ly - #s Ser Leu Glu Gln Ile
115 - # 120 - # 125
- - Trp Asn Asn Met Thr Trp Met Glu Trp Asp Ar - #g Glu Ile Asn Asn Tyr
130 - # 135 - # 140
- - Thr Ser Leu Ile His Ser Leu Ile Glu Glu Se - #r Gln Asn Gln Gln Glu
145 1 - #50 1 - #55 1 -
#60
- - Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Ly - #s Trp Ala Ser Leu
Trp
165 - # 170 - # 175
- - - - (2) INFORMATION FOR SEQ ID NO:9:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
- - GATCCATGGG TGCGAGAGCG TCAGTATTAA GCGGGGGAGA ATTAGAT - #
47
- - - - (2) INFORMATION FOR SEQ ID NO:10:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
- - CGATCTAATT CTCCCCCGCT TAATACTGAC GCTCTCGCAC CCATG - #
- #45
- - - - (2) INFORMATION FOR SEQ ID NO:11:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
- - GATCTCTAAG GATCCTTA - # - #
- # 18
- - - - (2) INFORMATION FOR SEQ ID NO:12:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
- - GATCTAAGGA TCCTTAGA - # - #
- # 18
- - - - (2) INFORMATION FOR SEQ ID NO:13:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
- - CAGATCTCCG GAGTAGCACC CACC - # - #
24
- - - - (2) INFORMATION FOR SEQ ID NO:14:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
- - GAGATCTGTT AAGCATTCCA AGGCAC - # - #
26
- - - - (2) INFORMATION FOR SEQ ID NO:15:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
- - CTCGAAGATC TCCAGGGCTA TTGAGGCGCA - # - #
30
- - - - (2) INFORMATION FOR SEQ ID NO:16:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
- - CTCGAAGATC TATTACCACA AACTTGCCCA - # - #
30
- - - - (2) INFORMATION FOR SEQ ID NO:17:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
- - CGCTGATCAA TGAGAGTGHA GGAGAAATAT CAGC - # -
# 34
- - - - (2) INFORMATION FOR SEQ ID NO:18:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
- - CGCTGATCAT TATAGCAAAA TCCTTTCCAA GCCC - # -
# 34
- - - - (2) INFORMATION FOR SEQ ID NO:19:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 357 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
- - GGAGTAGCAC CCACCAAGGC AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AG -
#CAGTGGGA 60
- - ATAGGAGCTT TGTTCCTTGG GTTCTTGGGA GCAGCAGGAA GCACTATGGG CG -
#CAGCGTCA 120
- - ATGACGCTGA CGGTACAGGC CAGACAATTA TTGTCTGGTA TAGTGCAGCA GC -
#AGAACAAT 180
- - TTGCTGAGGG CTATTGAGGC GCAACAGCAT CTGTTGCAAC TCACAGTCTG GG -
#GCATCAAG 240
- - CAGCTCCAGG CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GC -
#TCCTGGGG 300
- - ATTTGGGGTT GCTCTGGAAA ACTCATTTGC ACCACTGCTG TGCCTTGGAA TG - #CTTAA
357
- - - - (2) INFORMATION FOR SEQ ID NO:20:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 345 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
- - AGGGCTATTG AGGCGCAACA GCATCTGTTG CAACTCACAG TCTGGGGCAT CA -
#AGCAGCTC 60
- - CAGGCAAGAA TCCTGGCTGT GGAAAGATAC CTAAAGGATC AACAGCTCCT GG -
#GGATTTGG 120
- - GGTTGCTCTG GAAAACTCAT TTGCACCACT GCTGTGCCTT GGAATGCTAG TT -
#GGAGTAAT 180
- - AAATCTCTGG AACAGATTTG GAATAACATG ACCTGGATGG AGTGGGACAG AG -
#AAATTAAC 240
- - AATTACACAA GCTTAATACA CTCCTTAATT GAAGAATCGC AAAACCAGCA AG -
#AAAAGAAT 300
- - GAACAAGAAT TATTGGAATT AGATAAATGG GCAAGTTTGT GGTAA - #
345
- - - - (2) INFORMATION FOR SEQ ID NO:21:
- - (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 531 base - #pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
- - (ii) MOLECULE TYPE: cDNA
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
- - GGAGTAGCAC CCACCAAGGC AAAGAGAAGA GTGGTGCAGA GAGAAAAAAG AG -
#CAGTGGGA 60
- - ATAGGAGCTT TGTTCCTTGG GTTCTTGGGA GCAGCAGGAA GCACTATGGG CG -
#CAGCGTCA 120
- - ATGACGCTGA CGGTACAGGC CAGACAATTA TTGTCTGGTA TAGTGCAGCA GC -
#AGAACAAT 180
- - TTGCTGAGGG CTATTGAGGC GCAACAGCAT CTGTTGCAAC TCACAGTCTG GG -
#GCATCAAG 240
- - CAGCTCCAGG CAAGAATCCT GGCTGTGGAA AGATACCTAA AGGATCAACA GC -
#TCCTGGGG 300
- - ATTTGGGGTT GCTCTGGAAA ACTCATTTGC ACCACTGCTG TGCCTTGGAA TG -
#CTAGTTGG 360
- - AGTAATAAAT CTCTGGAACA GATTTGGAAT AACATGACCT GGATGGAGTG GG -
#ACAGAGAA 420
- - ATTAACAATT ACACAAGCTT AATACACTCC TTAATTGAAG AATCGCAAAA CC -
#AGCAAGAA 480
- - AAGAATGAAC AAGAATTATT GGAATTAGAT AAATGGGCAA GTTTGTGGTA A - #
531
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